PROJECT DESCRIPTION We propose to define the reproductive functions of 14 novel testis-specific secreted or transmembrane proteins using newly developed and rapid CRISPR/Cas9 gene manipulation strategies. Over the last two decades, the Matzuk laboratory at Baylor College of Medicine and the Ikawa and Okabe laboratories at Osaka University, world leading experts in CRISPR/Cas9 technology, have succeeded in producing >200 mouse models to study reproductive processes in vivo. In this proposal, we will bring together our expertise in bioinformatics and manipulation of the mouse genome to characterize the in vivo functions and mechanisms of action of 14 novel testis-specific proteins. Using bioinformatics strategies, the Matzuk laboratory identified over 100 genes that are specifically expressed in mouse testis, that previously had not been functionally characterized in vitro or in vivo, and that have human orthologs. In parallel, the Ikawa and Okabe laboratories have developed the CRISPR/Cas9 system to rapidly and efficiently mutate genes in vivo, thereby avoiding the embryonic stem (ES) cell and chimera stages that have proved the most troublesome and labor-intensive for the traditional generation of knockout mice. Using our synergistic approaches, our laboratories are focusing on the functional characterization of 2 novel genes that encode secreted ligands and 12 novel genes that encode potential transmembrane proteins. These 14 proteins were chosen because of their potential relevance to infertility in men and their likelihood as druggable targets for male contraception; indeed, 70% of the FDA-approved drugs target either transmembrane or secreted proteins. Therefore, we have carefully selected our genes to not only have potential relevance to infertility in men but also as future potential targets for small molecules that specifically inhibit spermiogenesis, sperm morphogenesis, sperm motility, and/or fertilization. To date, we have evaluated the fertility status of mice with mutations in 12 of the 14 novel genes and discovered that null mutations in 6 genes lead to male sterility secondary to sperm morphogenesis, motility, or fertilization defects, 1 null mutation results in severe subfertility, 1 null mutation leads to an in vitro fertilization defect, and 4 null mutations did not alter fertility. Thus, mutations in the majority of our identified novel genes lead to defects in sperm formation or function, areas in which our groups have abundant skills in functional analysis and for which we have published over 100 papers. Working together, our groups will dedicate the first year of our proposal to defining the fertility phenotypes of the mutant mouse models and will devote the remaining four years to mechanistically characterizing only the proteins deemed to be essential for male reproduction and having relevance to contraception. These proof-of-principle studies have important translational implications for human reproductive genetics and contraceptive development.